Chimeric fibroblast growth factors

ABSTRACT

The present invention relates to novel chimeric fibroblast growth factors (FGF) wherein the alanine at amino acid 3 and serine 5 of native human recombinant basic fibroblast growth factor are replaced with glutamic acid. The N-terminus sequence of the present chimeric FGFs identify homology with that of human acidic fibroblast growth factor. The mitogenic properties of the native human recombinant basic FGF are exhibited by the present chimeric FGFs, and they are efficiently expressed in E. coli at significantly greater yields that previously reported. Novel variants of this new glu 3 ,5 basic fibroblast growth factor, such as those in which cysteine 78 and cysteine 96 are replaced, e.g., with serine or other amino acids, to produce stabilized versions of the glu 3 ,5 basic FGF and eliminate disulfide scrambled forms, are also described.

This is a continuation-in-part of copending application(s) Ser. No.07/615,202 filed on Nov. 23, 1990, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to novel chimeric basic fibroblast growthfactors and to the enhanced production of such factors (bFGF).

Polypeptide growth factors are hormone-like modulators of cellproliferation and differentiation. Growth factors are responsible forthe regulation of a variety of physiological processes, includingdevelopment, regeneration and wound repair.

In the course of study of these factors, a number have been identifiedon the basis of the ability of extracts from various tissues, such asbrain, pituitary and hypothalamus, to stimulate the mitosis of culturedcells. Numerous shorthand names have been applied to active factors inthese extracts, including epidermal growth factor, platelet-derivedgrowth factor, nerve growth factor, hematopoietic growth factor andfibroblast growth factor.

Fibroblast growth factor (FGF) was first described by Gospodarowicz in1974 (Nature 249: 123-127) as derived from bovine brain or pituitarytissue which was mitogenic for fibroblasts and endothelial cells. It waslater noted that the primary mitogen from brain was different from thatisolated from pituitary. These two factors were named acidic and basicFGF, respectively, because they had similar if not identical biologicalactivities but differed in their isolectric points. Acidic and basicfibroblast growth factors (recently reviewed by Burgess, W. H., andMaciag, T. Ann. Rev. Biochem. 58: 575-606 (1989)) appear to be normalmembers of a family of heparin-binding growth factors that influence thegeneral proliferation capacity of a majority of mesoderm- andneuroectoderm-derived cells (Gospodarowicz, D., et al., Nat. CancerInst. Mon. 48:109-130 (1978)), including endothelial cells, smoothmuscle cells, adrenal cortex cells, prostatic and retina epithelialcells, oligodendrocytes, astrocytes, chrondocytes, myoblasts andosteoblasts (Burgess and Maciag, cited above at page 584). Althoughhuman melanocytes respond to the mitogenic influences of basicfibroblast growth factor but not acidic FGF most avian and mammaliancell types respond to both polypeptides (ibid.).

In addition to eliciting a mitogenic response that stimulates cellgrowth, fibroblast growth factors can stimulate a large number of celltypes to respond in a non-mitogenic manner. These activities includepromotion of cell migration into wound areas (chemotaxis), initiation ofnew blood vessel formation (angiogenesis), modulation of nerveregeneration (neurotropism), and stimulation or suppression of specificcellular protein expression, extracellular matrix production and cellsurvival important in the healing process (Burgess and Maciag, citedabove, pages 584 to 588).

These properties, together with cell growth promoting action, provide abasis for using fibroblast growth factors in therapeutic approaches toaccelerate wound healing and in prevention and therapeutic applicationsfor thrombosis, artheriosclerosis, and the like. Thus, fibroblast growthfactors have been suggested to promote the healing of tissue subjectedto trauma (Davidson, J. M., et al. J. Cell Bio. 100:1219-1227 (1985)),to minimize myocardium damage in heart disease and surgery (U.S. Pat.Nos. 4,296,100 and 4,378,347 to Franco), and to increase neuronalsurvival and neurite extension (Walicke, P., et al., Proc. Nat. Acad.Sci. USA 83: 3012-3016 (1986)).

Complementary DNA clones encoding human acidic and human and bovinebasic fibroblast growth factors have been isolated and sequenced, andthe predicted amino acid sequences derived from the complementary DNAsagree with the structures determined by protein sequence analysis(summarized by Burgess and Maciag, cited above, at pages 580-581). Thedata predict acidic fibroblast growth factor (hereafter referred to asaFGF) to have 155 amino acids (ibid). The gene for basic fibroblastgrowth factor (hereafter referred to as bFGF) also codes for a 155residue protein. For both aFGF and bFGF N-terminally truncated formsthat exhibit full biologic activity including a 146-amino acid bFGForiginally isolated and sequenced (Esch, F., et al, Proc. Nat. Acad.Sci. USA 82 6507-6511 (1985)) and a 131-amino acid form. Analysis of thestructures demonstrates a 55% identity between aFGF and bFGF (Burgessand Maciag, cited above at page 581).

Basic fibroblast growth factor may be extracted from mammalian tissue,but this requires several steps even when heparin-linked affinitychromatography is employed (U.S. Pat. Nos. 4,785,079 and 4,902,782 toGospodarowicz, et al.), and the 146-amino acid species is generallyobtained if extraction is done in the absence of protease inhibitors(ibid., column 9, lines 29 to 32). Bovine and human basic fibroblastgrowth factor cDNA have been expressed in E. coli (Iwane, M., et al.,Biochem. Biophys. Res. Commun. 146:470-477 (1987) and Squires, C. H., etal., J. Biol. Chem. 263:16297-16302 (1988)) and S. Cervisiae (Barr, P.J.. et al., J. Biol. Chem. 263: 16471-16478 (1988)). However, reportedyields of product are low (see Eur. Pat. Ap. Pub. No. 228,449 to Esch,et al., page 18), and recombinant factors exhibit a marked tendency toundergo thiol-disulfide interchanges promoted by free thiol groups inthe protein that result in the formation of disulfide scrambled species(Iwane, cited above).

A number of basic fibroblast growth factor analogues have beensuggested. Muteins of bFGF having amino or carboxyl terminal amino acidsdeleted, amino acids added, cysteine substituted with a neutral aminoacid such as serine, or aspartic acid, arginine, glycine, serine, orvaline substituted with other acids have been suggested to have enhancedstability (Eur. Pat. Ap. Pub. No. 281,822 to Seno, et al., page 4, lines1 to 3, and page 6, line 29 to page 7, line 19); the muteins comprisetwo or three additions, deletions or substitutions, with substitution ofserine for cysteine the most preferred substitution (page 7, lines 18 to23). Arakawa and Fox (Eur Pat. Ap. Pub. No. 320, 148) suggestedreplacing at least one, and more preferably two, of the cysteines foundin natural bFGF with a different amino acid residue to yield a morestable analogue (page 4, lines 44 to 47); serine was illustrated in theExamples (page 13, lines 22 to 23), but alanine, aspartic acid andasparagine were also suggested (page 5, line 26 and page 13, line 25).Similarly, recombinant aFGFs having extraneous bond-forming cysteinereplaced with serine, and oxidation-prone cysteine, methionine andtryptophan replaced with alanine, valine, leucine or isoleucine, toyield factors having enhanced or improved biological activity have alsobeen suggested (Eur. Pat. Ap. Pub. No. 319,052 to Thomas Jnr andLinemeyer, page 17, lines 8 to 20).

A bFGF mutein lacking 7 to 46 amino acids from the carboxyl terminusand, optionally, having amino acids replacements was suggested to haveimproved stability while retaining activity in Eur. Pat. Ap. Pub. No.326,907 to Seno, et al. (page 2, line 50 to page 3, line 4). Fiddes, etal, (Eur. Pat. Ap. Pub. No. 298723) suggested replacing basic orpositively charged residues in the heparin binding domain encompassingresidues 128 to 138 with neutral or negatively charged amino acids toproduce forms of FGF having reduced heparin binding ability and enhancedpotency (page 5, line 45, and page 5, line 54 to page 6, line 16).Bergonzoni, et al., suggested six analogues: 1) M1-bFGF, lackingresidues 27 to 32; M2-bFGF, lacking residues 54 to 58; M3-bFGF, lackingresidues 70 to 75; M4-bFGF, lacking residues 78 to 83; M5 -bFGF, lackingresidues 110 to 120; M5a-bFGF, having the position 128 lysine and theposition 129 arginine replaced with glutamine residues; and M6b-bFGF,having the positions 119 and 128 lysines and the positions 118 and 129arginines replaced by glutamine residues (Eur. Pat. Ap. Pub. No.363,675, column 6, line 48 to column 7, line 13).

However, new stable and active forms of fibroblast growth factors areincreasingly sought to use in the therapies indicated hereinabove.

SUMMARY OF INVENTION

The present invention relates to novel, full length (coding for 155amino acid) human basic fibroblast growth factor genes and proteinswhich have the alanine residue at position 3 and the serine residue atposition 5 of the native bFGF replaced with glutamic acid. Glu³,5 hbFGFof this invention hares sequence identity with human acidic FGF at theN-terminal 8 amino acids and can thus be considered a chimeric FGF. Morespecifically, these factors are of human FGF but other mammalian speciesof FGFs are available through the present invention.

The glu³,5 chimeric fibroblast growth factor has the mitogenicproperties of tissue-derived bFGF, but expression in E. coli issignificantly greater than the native sequence. Thus, this inventionprovides novel, biologically active FGF and a method of preparing it inhigh yield.

The same finding exists with respect to novel variants of glu³,5 hbFGFFor example, a stabilized version of the growth factor is prepared byreplacing cysteine 78 and cysteine 96 with amino acids that eliminatethiol-disulfide interchange (disulfide scrambling), such as serine.Thus, this invention not only modifies hbFGF (1-155) to significantlyincrease the yield of the factor expressed in E. coli, but alsofacilitates purification and enhances stability.

Therefore, this invention provides novel, biologically active fibroblastgrowth factors and methods of preparing these on a preparative scale. Ina preferred embodiment, DNA encoding novel FGF of this invention isinserted into plasmids or vectors, which may be conveniently andefficiently conserved, stored or transported, if desired. The plasmidsor vectors are then used to transform or transfect microorganism, e.g.,E coli. which likewise may be used to conserve, store, or transport, ifdesired, the genomic material encoding the novel FGF of this invention.Culture of these microorganisms under conditions that express thefactors yield the polypeptides in abundance.

Since, as described above, growth factors released into traumatizedareas accelerate the normal healing process, the novel fibroblast growthfactors of this invention have therapeutic applications for healingburns, surgical incisions, and other wounds; for treating skin ulcers,including bedsores and the like; for cardiovascular conditions andrestarting blood flow after heart attacks by revascularizing the damagedtissue; for enhancing bone repair and treating musculoskeletal injuries;and in neurodegenerative and other disease states.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1. Construction of Glu³,5,Ser⁷⁸,96 hbFGF cDNA. Using the expressionplasmid pT7 glu³,5 hbFGF prepared in Example 2 as a template, cys⁷⁸ toser⁷⁸ and cys⁹⁶ to ser⁹⁶ mutations are directed using the followingpolymerase chain reaction mixtures: (1) T7 sense plus ser⁷⁸ antisenseprimers; and (2) T7 antisense plus ser⁹⁶ sense primers. A polymerasechain reaction is then performed from (1) and (2) using T7 sense and T7antisense primers as described in Example 3.

FIG. 2. Heparin HPLC of Natural hbFGF. Bound hbFGF (containing 154 aminoacids) is eluted from a heparin sepharose column using a linear 0.6 to3.0M NaCl gradient at 0.7 ml/min and monitored at 280 nm as described inExample 5.

FIG. 3. Heparin HPLC of Reduced Recombinant hbFGF. An elution profilefrom heparin HPLC of a portion of pooled material from heparin sepharosechromatography that has been reduced with dithiotheitol as described inExample 6.

FIG. 4. Reverse Phase HPLC of Glu³,5,Ser⁷⁸,96 hbFGF. A sample fromheparin HPLC (8 μg) is loaded onto a 0.45×25 cm Vydac C₄ column andeluted at 0.7 ml/min using a 0.1% trifluoroacetic acid/acetonitrilesolvent system (0 to 28% acetonitrile in 15 min, 28 to 60% in 99 min,and 60 to 80% in 10 min) as described in Example 6.

FIG. 5. Bioassay Comparison of native bFGF with Recombinant bFGF's. Themitogenic activity of bFGF isolated from bovine brain is compared withhuman recombinant bFGF's on the proliferation of aortic arch bovinevascular endothelial cells as described in Example 7. Cells are grown inthe presence of different amounts of bovine brain bFGF (10-155) (- -);natural sequence recombinant hbFGF (- -); and glu³,5 hbFGF (-o-)(determined by amino acids analysis) as indicated. After 4 days, acidphosphatase activity , equivalent to cell number over the cell densityrange examined, is determined at 405 nm.

FIG. 6. Bioassay Comparison of native bFGF with Chimeric bFGF's. Themitogenic activity of bovine brain bFGF (10-155) (-o-) andglu³,5,ser⁷⁸,96 hbFGF (- -) is compared using cells maintained in thepresence of different amounts (determined by amino acids analysis) ofgrowth factors as indicated for 5 days and cell number determined asdescribed in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the enhanced production andstabilization of new, basic fibroblast growth factors having about 155amino acids. Although the human recombinant example provided herein, theFGF of the present invention is applicable to other mammalian species. Anovel recombinant hbFGF of this invention is prepared by replacingalanine 3 and serine 5 with glutamic acid in full length human basicfibroblast growth factor. Glu³,5 hbFGF shares sequence identity withhuman aFGF at the N-terminal 8 amino acids and is thus considered achimeric analogue of haFGF and hbFGF. This mutation significantlyincreases protein yield expressed in Escherichia coli as compared tonative sequence bFGF.

Both recombinant native bFGF and glu³,5 hbFGF exhibit microheterogeneityon heparin- and reverse phase- high performance liquid chromatography.While not wishing to be bound to any theory, this microheterogeneityappears to be due to thiol-disulfide interchange (disulfide scrambling)because it can be eliminated by treatment of the growth factors with areducing agent prior to chromatography. Generation of a stabilizedversion of the growth factor and elimination of disulfide scrambledforms is accomplished by replacement of cysteine 78 and cysteine 96 withserine by site-directed mutagenesis.

A protein is defined herein as basic FGF if it shows FGF activity in invitro and in vivo assays (summarized by Burgess and Maciag, cited above,pages 584 to 586); binds to heparin; and is eluted from heparinsepharose at 1.5-1.7M NaCl; and reacts immunologically with antibodiesprepared using human or bovine basic FGF or synthetic or native peptidesthereof, or to synthetic analogues of bFGF sequences conjugated tobovine serum albumin. A protein is defined herein as acidic FGF if itshows FGF activity in in vitro and in vivo assays; binds to heparin; andis eluted at 1.0-1.2M NaCl from heparin sepharose; and isimmunologically reactive with antibodies prepared against human orbovine aFGF or against synthetic or native peptides thereof. A chimericfibroblast growth factor shares the sequence of both types. Any type ofmammalian fibroblast growth factor is encompassed by this invention,particularly human fibroblast growth factor.

The chimeric fibroblast growth factors of this invention include glu³,5hbFGF and glu³,5,ser⁷⁸,96 hbFGF having about 155 amino acids, andtruncated forms having about 154 amino acids (e.g., those having noN-terminal methionine; see Example 6 (b)). This invention alsoencompasses glu³,5 hbFGF analogues having the cysteine residues atpositions 78 and 96 replaced with other amino acids, such as, forexample alanine, glycine, arginine, tryptophan, lysine, aspartic acid,glutamic acid, asparagine, glutamine, histidine, isoleucine, leucine,valine, phenylalanine, tyrosine, methionine, serine, threonine orproline. Moreover, the FGF derivatives of this invention are not speciesspecific, and include, for example, bovine FGF counterparts and othersthat share similar sequence homology with hbFGF.

The novel fibroblast growth factors of this invention may be prepared byassembling polypeptides from constituent amino acids, or from aminoacids or peptides and polypeptides, using chemical or biochemical meansknown to those skilled in the art, such as, for example, by adding aminoacids sequentially to shorter fibroblast forms at the N-terminus.Alternatively, the novel fibroblast growth factors of this invention maybe prepared by recombinant protein synthesis involving preparation ofDNA encoding chimeric FGF, insertion of the DNA into a vector,expression of the vector in host cells, and isolation of the recombinantFGF thereby produced.

DNA encoding the FGF of this invention may be prepared by altering agene of human or bovine basic fibroblast growth factor by nucleotidedeletions, nucleotide additions, or point mutations produced usingstandard means. An illustration is set out in Example 1. Because of thedegeneracy of the genetic code, a variety of codon change combinationscan be selected to form DNA that encodes the FGF of this invention, sothat any nucleotide deletion(s), addition(s), or point mutation(s) thatresult in a DNA encoding chimeric FGF are encompassed by this invention.Since certain codons are more efficient for polypeptide expression incertain types of organisms, the selection of fibroblast gene alterationsto yield DNA material that codes for the FGF of this invention arepreferably those that yield the most efficient expression in the type oforganism which is to serve as the host of the recombinant vector.Altered codon selection may also depend upon vector constructionconsiderations.

Fibroblast growth factor DNA starting material which can be altered toform chimeric DNA may be natural, recombinant or synthetic. Thus, DNAstarting material may be isolated from tissue or tissue culture,constructed from oligonucleotides using conventional methods, obtainedcommercially, or prepared by isolating RNA coding for bFGF fromfibroblasts, using this RNA to synthesize single-stranded cDNA which canbe used as a template to synthesize the corresponding double strandedDNA.

Illustrating the present invention are cloned complementary DNAsequences defining chimeric fibroblast polypeptide sequences such asthat constructed in Examples 2 and 3. Also encompassed are DNA sequenceshomologous or closely related to complementary DNA described herein,namely DNA sequences which hybridize, particularly under stringentconditions, to chimeric fibroblast cDNA, and RNA corresponding thereto.In addition to the chimeric FGF-encoding sequences, DNA encompassed bythis invention may contain additional sequences, depending upon vectorconstruction sequences, that facilitate expression of the gene.

DNA encoding the chimeric growth factors of this invention, or RNAcorresponding thereto, are then inserted into a vector, e.g., a pBR,pUC, pUB or pET series plasmid, and the recombinant vector used totransform a microbial host organisms. Host organisms may be bacterial(e.g., E. coli or E. subtilis), yeast (e.g., S. cervisiae or mammalian(e.g., mouse fibroblast). This invention thus also provides novel,biologically functional viral and circular plasmid RNA and DNA vectorsincorporating RNA and DNA sequences describing the chimeric growthfactors generated by standard means. Culture of host organisms stablytransformed or transfected with such vectors under conditionsfacilitative of large scale expression of the exogenous, vector-borneDNA or RNA sequences and isolation of the desired polypeptides from thegrowth medium, cellular lysates, or cellular membrane fractions yieldsthe desired products. An example of expression of hbFGF mutants in E.coli is given in Example 4.

The present invention provides for the total and/or partial manufactureof DNA sequences coding for glu³,5 hbFGF, glu³,5, ser⁷⁸,96 hbFGF, andother glu³,5 hbFGF having the cysteines at positions 78 and 96 replacedwith other amino acids that eliminate disulfide scrambling, andincluding such advantageous characteristics as incorporation of codonspreferred for expression by selected non-mammalian hosts, provision ofsites of cleavage by restriction by endonuclease enzymes, and provisionof additional initial, terminal or intermediate DNA sequences whichfacilitate construction of readily expressed vectors. Correspondingly,the present invention provides for manufacture (and development by sitespecific mutagenesis of cDNA and genomic DNA) of DNA sequences codingfor microbial expression of chimeric fibroblast growth factor whichdiffer from the forms specifically described herein in terms of identityor location of one or more amino acids residues (i.e., deletionanalogues containing less than all of the residues specified for hbFGF,and/or substitution analogues wherein one or more residues are replacedby and/or addition analogues wherein one or more residues are added to aterminal or medial portion of the polypeptide), and which share thebiological properties of glu³,5 hbFGF and glu³,5, ser⁷⁸,96 hbFGF.

DNA (and RNA) sequences of this invention code for all sequences usefulin securing expression in procaryotic or eucaryotic host cells ofpolypeptide products having at least a part of the primary structuralconformation, and one or more of the biological properties of chimericfibroblast growth factor which are comprehended by: (a) the DNAsequenced encoding glu³,5 hbFGF and glu³,5,ser⁷⁸,96 hbFGF as describedherein, or complementary strands; (b) DNA sequences which hybridize(under hybridization conditions as described herein or more stringentconditions) to DNA sequences defined in (a) or fragments thereof; and(c) DNA sequences which, but for the degeneracy of the genetic code,would hybridize to the DNA sequenced defined in (a) and (b) above.Specifically comprehended are genomic DNA sequenced encoding allelicvariant forms of chimeric fibroblast growth factors included therein,and sequences encoding chimeric fibroblast growth factor RNA, fragmentsthereof, and analogues wherein RNA or DNA sequenced may incorporatecodon facilitating transcription or RNA replication of messenger RNA innon-vertebrate hosts.

Isolation and purification of microbially expressed polypeptidesprovided by the invention may be by conventional means including, forexample, preparative chromatographic separations such as thatillustrated in FIGS. 2 and 3, and immunological separations, includingmonoclonal and/or polyclonal antibody preparations, i.e. examplepurification is given in Example 5.

As summarized above and described in detail in the Examples below, andexample chimeric fibroblast growth factor of this invention is fulllength (155 amino acids) human recombinant basic fibroblast growthfactor having alanine 3 and serine 5 replaced with glutamic acidexpressed, using the T7 RNA polymerase expression system, in E. coli.(The numbering for bFGF adopted here is for the 155 amino acid form asdescribed in Abraham et al, 1986 and refers to the methionine codon asposition 1) Both recombinant native bFGF and glu³,5 hbFGF exhibitextensive microheterogeneity on heparin- and RP-PHLC (FIG. 2) which iseliminated by treatment of the growth factor with a reducing agent suchas dithiothreitol prior to chromatography (FIG. 3). Generation of astabilized version of the growth factor and elimination of disulfidescrambled forms is accomplished by replacement of cysteine 78 andcysteine 96 with serine by site-directed mutagenesis (Example 3).

The yield of both glu³,5 hbFGF and glu³,5,ser⁷⁸,96 hbFGF in thisexpression system, like aFGF cDNA, is 10-fold higher than parental bFGFcDNA (Example 5). Gu³,5 hbFGF and glu³,5,ser⁷⁸,96 hbFGF share sequenceidentity with haFGF at the N-terminal 8 amino acids. Thus, thesederivatives are chimeric.

The polypeptides of this invention retain biological activity asfibroblast growth factors. For example when the mitogenic properties ofrecombinant hFGF and mutant proteins are compared to bFGF (10-155)originally isolated from bovine brain (Example 7), human recombinantbFGF and glu³,5 hbFGF show a dose-dependent stimulation of endothelialcell growth that was essentially identical to that for bovine brain bFGF(FIG. 5). Replacement of cysteine 78 and 96 with serine to giveglu³,5,ser⁷⁸,96 hbFGF had no effect on the mitogenic potency and gave adose-response curve that was indistinguishable from that determined fortissue-derived bovine bFGF (FIG. 6). Sequence I.D. Numbers 1 and 2disclose the sequences of glu³,5 hbFGF and glu³,5, Ser⁷⁸,96 hbFGF,respectively.

The modifications to hbFGF described herein significantly increase theyield of growth factor expressed, facilitate its purification,eliminates microheterogeniety due to disulfide scrambling, and enhancesstability while retaining full biological activity.

EXAMPLES

The following examples are presented to further describe and explain thepresent invention and the characterization techniques employed, andshould not be taken as limiting in any regard. Unless otherwiseindicated, all parts and percentages are by weight, and are based on theweight at the particular stage of the processing being described.

EXAMPLE 1 Construction of an Expression Plasmid

A synthetic gene encoding the 155 amino acid form of human bFGF(Abraham, J. A., et al, EMBO J. 5: 2523-2528 (1986)) cloned into pUC 18was purchased from British Bio-technology, Oxford, UK. Destruction ofthe internal Nco1 restriction site at positions -2 to 3, which includesthe N-terminal methionine codon of the bFGF cDNA, and introduction of aunique Nde1 site is as follows. The nucleotide sequence (-12 to 36) tobe changed (a, below) is excised from pUC 18 with HindIII and BspMII anda synthetic fragment (b, below) containing an internal Nde1 site clonedinto pUC 18. This cloning results in a construct that contains a 4nucleotide deletion in the upstream non-coding region compared to theoriginal construct (see below). This deletion has no effect on therelative protein yields of bFGF using the expression system describedbelow.

5' AGCTTACCTGCCATGGCAGCCGGGAGCATCACCACGCTGCCCGCCCTT 3' (a)

5' AGCTTCATATGGCAGCCGGGAGCATCACCACGCTGCCCGCCCTT 3' (b)

Only the sense strands are shown for the original (a) and modified (b)fragments, respectively. The codon underlined indicates the position ofthe methionine start codon.

The cDNA encoding bFGF is then excised from pUC 18 with Nde1 and BamH1and cloned into the Nde1 and BamH1 sites of the expression vector pT7Kan 5, derivative of pET-3a (plasmid for Expression by bacteriophage T7,as defined in Rosenberg, A., et al, Gene 56: 125-135 (1987) at page 128)containing the T7 promoter for RNA polymerase.

EXAMPLE 2 Construction of Glu³,5 hbFGF

The protocol for the construction of glu³,5 hbFGF is identical to thatdescribed above for the introduction of the Nde1 restriction site exceptthat the region encoding the first 5-terminal amino acids of basic FGF(c) are changed to encode those of acidic FGF (d):

5' AGCTTCATATGGCAGCCGGGAGCATCACCACGCTGCCCGCCCTT 3' (c)

5' AGCTTCATATGGCTGAAGGGGAAATCACCACGCTGCCCGCCCTT 3' (d)

Only the sense strands are shown for the original (c) and modified (d)fragments, respectively. The codons underlined indicate those changed toencode glutamic acid at positions 3 and 5.

EXAMPLE 3 Construction of Glu³,5 Ser⁷⁸,96 hbFGF

The expression plasmid pT7 glu³,5 hbFGF is used as a template foroligonucleotide site-directed mutagenesis. Two mutagenic oligonucleotideprimers are designed to change cysteine codons at positions 78 and 96 toserine codons. The primer for serine at position 96 is to the sensestrand (60-mer; 238-297) whereas that for serine at position 78 is tothe anti-sense strand (30-mer; 251-222). In addition to these mutagenicprimers, primers to the T7 promotor (nucleotide -12 to +13) andterminator regions (nucleotide -75 to -54) are designed (19).

Mutation of the modified FGF gene is accomplished by use of a polymerasechain reaction (PCR). Two reaction mixtures containing HindIII cutplasmid DNA are prepared as shown schematically in FIG. 1: (i) T7 senseplus Ser 78 antisense primers to yield an expected 319 base pairproduct, and (ii) T7 antisense plus Ser 96 sense primers to produce anexpected 294 basepair product. PCR mixtures are prepared according tothe manufacturer's instructions (Perkin Elmer Cetus, Norwalk, CT). PCRis performed using Taq polymerase for 30 amplification cycles each 92°C. for 1 min, 50° C. for 5 sec, and 72° C. for 1 min, and the productsanalyzed by agarose gel electrophoresis.

Excess primers are separated from the amplified DNA fragments by 3successive rounds of concentration and dialysis using 30,000 molecularweight Millipore microconcentrators. Portions of the retentates arecombined and amplified using the PCR as described above except that theprimers used correspond to the T7 promoter (sense) and T7 terminator(antisense) regions. See FIG. 1. The 599 basepair PCR product is thentreated with NdeI and BamHI and purified by agarose gel electrophoresis.The purified fragment is then cloned into the T7 expression vector,pET-3a(M13), a derivative of pET-3a.

EXAMPLE 4 Expression of Natural Sequence hbFGF and hbFGF Mutants

Following sequence verification (Sanger, F., et al., Proc. Nat. Acad.Sci. 74:5463-5467 (1977)), the genes encoding the bFGF mutants aretransformed into competent BL21 pLysS cells. E. coli cells harboring theplasmids are grown in Luria broth containing kanamycin sulfate (50 μg/mlor ampicillin (100 μg/ml) for plasmid glu³,5 ser⁷⁸,96 hbFGF andchloramphenicol (34 μg/ml) at 37° C. to about 0.6 absorbance units at600 nm. bFGF synthesis is induced by addition ofisopropyl-beta-D-thiogalactopyranoside (1 mM). Two hours postinduction,the cells are then harvested by centrifugation at 4° C.

EXAMPLE 5 Purification of hbFGF Mutants

Cell pellets from 1 liter cultures are resuspended in 30 ml 50 mM Tris,0.1 mM EDTA buffer, pH 7.6, and lysed by 3 rapid freeze/thaw cycles. Thelysate is then treated with DNase I (20 μg/ml) in the presence of 5 mMMgCl₂ for 20 min at 4° C. and centrifuged at 10,000 ×g for 20 min toremove cell debris. bFGF activity is found to be equally distributed inthe pellet and supernatant fractions.

FGF is purified from the supernatant solution by heparin-sepharosecolumn chromatography as described by Gospodarowicz, D., et al., Proc.Nat. Acad. Sci. USA 81:6963-6967 (1984), and eluting with a linear saltgradient from 0.6 to 3.0M NaCl. The fractions containing growth factorare pooled and diluted with 10 mM, pH 7.6 Tris buffer to give a finalNaCl concentration of about 0.6M.

This is loaded onto a 0.75×7.5 cm TSK Heparin-5PW column (TosoHaas,Philadelphia, PA) equilibrated with 10 mM, pH 7.6 Tris, 0.6M NaCl.Elution of bound material is monitored at 280 nm and is accomplishedusing a linear salt gradient (0.6 to 3.0M NaCl in 60 min) at a flow rateof 0.7 ml/min.

Using the T7 expression system described in Example 4, the yield ofnative sequence hbFGF (2-155) is about 0.8 mg/1 bacterial culture. Withnative sequence haFGF, a 6 to 8 mg/liter yield is obtained. Glu³,5 hbFGFexpressed in the same system gives 8 to 10 mg/liter of purified factor.

Large scale fermentation (10L) of E. coli containing the plasmid pT7glu³,5,ser⁷⁸,96 gives about 1 mg of purified growth factor per g of cellpaste. Protein yield of the chimeric ser⁷⁸,96 variant and distributionof the protein in the supernatant and pellet fractions of the bacterialextract are similar to that observed for glu³,5 hbFGF.

EXAMPLE 6 Characterization of hbFGF and hbFGF Mutants (a)Chromatographic Behavior

Heparin HPLC purified bFGF's are analyzed by reverse phase highperformance liquid chromatography, RP-HPLC (C₄, Vydac, the separationsGroup, Hesperia, CA) using a 0.1% trifluoroacetic acid/acetonitrilegradient (0 to 28% CH₃ CN in 15 min, 28-60% in 99 min, and 60 to 30% in10 min) at a flow rate of 0.7 ml/min. Elution of bound material ismonitored at 210 nm.

The elution profile from heparin sepharose chromatography of a crudecell homogenate containing native sequence hbFGF (2-155) shows two majorprotein peaks both of which possess mitogenic activity and contain amajor protein species of M_(r) 17,000 by sodium dodeoyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). C₄ reverse phase-highperformance liquid chromatorgaphy (RP-HPLC) of material from each of thetwo peaks obtained from the heparin sepharose step followed byN-terminal sequence analysis of the resolved components identify atleast 3 distinct forms of bFGF.

As a first approach to analyze this apparent microheterogenetity, thecontribution of thiol-disulfide interchange (disulfide scrambling) inthe generation of chromatographically distinct species is assessed bytreatment with a reducing agent. Incubation of a portion of heparinsepharose purified bFGF with dithiothreitol (2 mM) for 10 min at 37° C.followed by RP-HPLC analysis shows that the peaks previously identifiedas bFGF species chromatograph essentially as a single peak.

High resolution TSK Heparin HPLC of 2 protein peaks containing FGF fromthe heparin sepharose step reveals 4 major protein components that eluteover a range of 1.6 to 2.3M NaCl (FIG. 2). Analysis by SDS-PAGE of thesepeaks shows P-I, P-III and P-IV to contain a single protein band thatmigrates with an M_(r) of 17,000 consistent with that hbFGF(2-155),whereas PII exhibits an M_(r) of about 22,000 and is identified byN-terminal sequence analysis as a contaminant. Treatment of a portion ofthe pooled material from heparin sepharose chromatography withdithiothreitol (5 mM) for 10 min at room temperature followed by heparinHPLC shows an increase in the amount of P-I, a reduction in that ofP-III and the disappearance of P-IV; the position and intensity of P-IIcontaining the Mr 22,000 impurity is unaffected by this treatment (FIG.3).

The chromatographic behavior of the glu³,5 hbFGF in the presence andabsence of dithiothreitol on heparin and RP-HPLC is similar to thatobserved for native sequence hbFGF. The cysteine to serine mutationgreatly facilitates the purification of this analogue since it behavesas a single species on heparin- and C₄ RP-HPLC (FIG. 4) and thuseliminates the need for dithiothreitol treatment during purification.

(b) Sequence Analyses

N-terminal sequence analyses of reverse phase purified proteins areperformed on a model 477A pulsed-liquid phase sequencer (from AppliedBiosystems, CA) equipped with an on-line phenylthiohydantoin- amino acidanalyzer (Model 120A, Applied Biosystems, CA). Amino acid compositionsare determined after HC1 gas phase hydrolysis (5.7M HC1/0.1% phenol; 24h at 110° C.) using a model 420A phenylisothiocyanate-derivatizerequipped with an on-line model 130A separation system (AppliedBiosystems, CA).

N-terminal sequence analysis of the material

isolated from heparin HPLC gives a single sequence consistent withglu³,5 hbFGF (2-155) indicating complete removal of the N-terminalmethionine.

(c) Molecular Weights

Molecular weight determinations are performed on a 10 to 15% gradientand 20% homogeneous polyacrylamide gels in the presence of sodiumdodecyl sulfate (SDS-PAGE) using a silver stain detection system(Phastgel System, Pharmacia/LKB).

hbFGF (2-155) migrates with an M_(r) of 17,000 compared to an M_(r)value of about 19,000 for glu³,5 hbFGF. Molecular weights calculatedfrom amino acid sequence data for hbFGF and the chimeric version are17,124 and 7,224, respectively. To resolve the apparent molecular weightdiscrepancy, a sample of glu³,5 hbFGF is analyzed by liquid-secondaryion mass spectrometry and gives a molecular ion of mass 17,365. Thisvalue is, within experimental error, consistent with that predicted fromsequence data. While not wishing to be bound to any theory, theanomalous migration of glu³,5 on polyacrylamide gels under denaturingconditions is most likely due to interference of protein-SDSinteractions from the glutamyl side chains at positions 3 and 5.

On SDS-PAGE glu³,5,ser⁷⁸,96 hbFGF also migrates as an M_(r) 19,000protein and not as a predicted M_(r) 17,000 species. This observation isconsistent with the aberrant migration noted for glu³,5 hbFGF.

EXAMPLE 7 Bioassay of Native and Mutant hbFGF Derivatives

The mitogenic activity of native sequence hbFGF and mutants isdetermined using bovine vascular endothelial cells derived from adultaortic arch as described by Esch, et al., Proc. Nat. Acad. Sci. USA 82:6507-6511 (1985). Cells are seeded at the initial density of 0.8×10⁴cells per 24-Well plate in 0.5 ml Dulbecco's modified Eagle's medium(DMEM) containing 10% calf serum (Hyclone, Logan, UT) supplemented withpenicillin (100 units/ml), streptomycin (100 μg/ml) and L-glutamine (2mM). Two hours after plating, 20 μl-aliquots of appropriate dilutions(0.001 to 100 ng/ml) of bFGF in DMEM containing 0.5% bovine serumalbumin (BSA) are added. After 5 days in culture, duplicate plates aretrypsinized and cell densities determined by cell counting in a Coultercounter.

Alternatively, growth curves in the presence and absence of bFGF aredetermined by measuring acid phosphatase levels after cell lysis usingp-nitrophenyl phosphate as substrate (Connolly, D. T., et al., Anal.Biochem. 152:136-140 (1986), page 137). Cells are seeded at an initialcell density of 1000 to 1200 cells per well (0.32 cm², 0.64 cm diameterflat bottomed 96 well plates) in 0.25 ml DMEM containing 10% calf serum,antibiotics and L-glutamine. After plating, 10 μl-aliquots ofappropriate dilutions of growth factor (0.001 to 100 ng/ml) in DMEMcontaining 0.5% BSA are added.

After 4 to 5 days in culture, each well is washed and 100 μl pH 5.5buffer containing 0.1M sodium acetate, 0.1% Triton X-100 and 10 mMp-nitrophenyl phosphate (Sigma 104 phosphatase substrate) are added toeach well. The plates are incubated at 37° C. for 2 hours, the reactionstopped by adding 10 μl of 1 N sodium hydroxide, and color developmentdetermined at 405 nm against a buffer blank incubated without cellsusing a UV max kinetic microplate reader (Molecular Devices, CA).Determinations are made in triplicate. Both methods giveindistinguishable dose-response curves.

When the mitogenic properties of recombinant hbFGF and mutant proteinsare compared to those of bFGF (10-155) originally isolated from bovinebrain, human recombinant bFGF and glu³,5 hbFGF show a dose-dependentstimulation of endothelial cell growth that is essentially identical tothat for bovine brain bFGF (FIG. 5) and exhibit doses for half-maximalstimulation (median effective dose, ED₅₀) of 0.3 to 1.0 ng/ml and amaximal stimulation between 3 and 10 ng/ml. Replacement of cysteine 78and 96 with serine to give glu³,5,ser⁷⁸,96 hbFGF has no effect on themitogenic potency and gives a dose-response curve that isindistinguishable from that determined for tissue-derived bovine bFGF(FIG. 6).

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention as defined in the appended claims.

The DNA sequences, plasmids and/or microorganisms deposited inconnection with the present patent application, except where specifiedto the contrary, are deposited in American Cyanamid Company's culturecollection maintained in Pearl River, N.Y. and are available to thepublic when legally appropriate to do so. Further, the following aredeposited additionally with the American Type Culture Collection (ATCC),12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. on the date indicatedwith the ATCC accession numbers indicated:

BL21 lysS/pET glu³,5 ser⁷⁸,96 deposited on Nov. 13, 1990 with ATCC No.68478

BL21 lys-S/pET glu³,5 hbFGF deposited on Nov. 13, 1990 with ATCC No.68477.

The above two contain the DNA of glu³,5 ser⁷⁸,96 hbFGF and glu³,5 hbFGFas described herein.

EXAMPLE 8 Derivatives with mEo-peg Compounds

Glu³,5 hbFGF (2-155) is prepared as described hereinabove. Polyethyleneglycol iodoacetates (meO-PEG-O₂ -CCH₂ I; MW=2000 and 5000) andiodoacetamide (MeO-PEG-NHCDCH₂ I; MW=5000) are prepared as describedherein.

Glu³,5 hbFGF (5mg/ml) in 1.0M Tris buffer pH8.6 containing 2mM EDTA isreduced by addition of dithiothreitol (5MM) and incurbated for 1h atroom temperature under an argon atomosphere. MeO-PEG-O₂ -CCH₂ I (MW=2000or =5000) MeO-PEG-NHCOCH₂ I (MW=5000) is added to give a finalconcentration of 25-50 mM and the reaction mixture isthen dialysedagainst phoshate buffered saline (PBS) at 4° C. for 12h.

EXAMPLE 9 Derivatized FGF

Glu³,5 hbFGF (5 mg/ml in 10 mM Tris buffer pH7.4 containing 1.5M NaC1 isreduced by addition of dithiothreitol (5 mM) and incubated for 0.5-1h atroom temperature under an argon atomosphere. Iodoacetic acid () 0.4M in1M Tris buffer ph8.5 ) is then added to give a final concentration of 50mM and the reaction mixture incubated in the dark for 2h at roomtemperature. The solution is then dialysed against 10 mM Tris buffer (pH7.0) containing 0.5M NaC1 for 12h.

Both the polyethylene glycol derivative of bFGF and thecarboxymethylated bFGF are assayed as described hereinabove.

EXAMPLE 10 Carboxymethylated FGF

Carboxymethylated bFGF: Treatment of Glu³,5 hbFGF (2-155) withiodoacetic acid under non-denaturing conditions results in thecarboxymethylation of 2 of the 4 cysteine residues of bFGF. Thepositions of modified cysteines are identified as cysteine 78 and 96 bypeptide mapping of a endoproteinase Glu-C digest of ¹⁴ C-labelledcarboxymethylated bFGF. Modification of cysteine 78 and 96 has no effecton the affinity of bFGF for heparin. The mitogenic activity and receptorbinding properties of Glu³,5 CMCys⁷⁸,96 hbFGF are indistinguishable fromthat of Glu³,5 hbFGF (FIG. 1) whereas fully carboxymethylated bFGF doesnot seem active. In contrast to unmodified bFGF, Glu³,5 hbFGF has ahalf-life at p 4 of about 5 min, whereas that for Glu³,5 CMCys⁷⁸,96hbFGF is >60 min (see FIG. 2).

Polyethylene glycol esters of bFGF: PEG-2000 and -5000 derivatives ofGlu³,5 hbFGF are fully active in bovine endothelial cell mitogenicassays (FIG. 3) and bind to heparin. PEG ester derivates undergohydrolysis to give carboxymethylated bFGF, a stabilized and fully activeanalog (see Example 8), which may be monitored by the appearance of an18Kd protein (glu³,5 CMcys⁷⁸,96 hbFGF) on sodium dodecyl sulfatepolyacrylamide gels.

Polyethylene glycol amide of bFGF: PEG-5000 derivative of glu³,5 hbFGFin contrast to the PEG esters of bFGF are stable. They are fully activein BalbC 3T3 fibroblast mitogenesis assays and compete as effectively asunmodified glu³,5 hbFGF in FGF receptor binding assays.

BIBLIOGRAPHY

1. Abraham, J. A., et al., EMBO J. 5: 2523-2528 (1986).

2. Arakawa, T. and Fox G. M., Eur. Pat. Ap. No. 320,148 (1989).

3. Barr, P. J., et al., Biol. chem. 263: 16471-16478 (1988).

4. Bergonzoni, L., et al., Eur. Pat. Ap. Pub. No. 363,675 (1989).

5. Burgess, W. H., and Maciag, T., Ann. Rev. Biochem. 8: 575-606 (1989).

6. Connolly, D. T., et al., Anal. Biochem. 152: 136-140 (1986).

7. Davidson, J. M., et al., J. Cell Bio. 100: 1219-1227 (1985).

8. Esch, F., et al., Proc. Nat. Acad. Sci. USA 82: 6507-6511 (1985).

9. Esch, F., et al., Eur. Pat. Ap. Pub. 228,449 (1986).

10. Fiddes, J. C., et al., Eur. Pat. Ap. Pub. No. 298,723 (1989).

11. Franco, W. P., U.S. Pat. No. 4,296,100 (1981).

12 Franco, W. P., U.S. Pat. No. 4,378,347 (1983).

13. Gospodarowicz. D., Nature 249:123-127 (1974).

14. Gospodarowicz. D., et al., Nat. Cancer Insti. Mon. 48 109-130(1978).

15. Gospodarowicz, D., et al., Proc. Nat. Acad. Sci. USA 81: 6963-6967(1984).

16. Gospodarowicz, D. et al., U.S. Pat No. 4,785,079 (1988).

17. Gospodarowicz. d. et al., U.S. Pat No. 4,902,782 (1990).

18. Iwane, M., et al., Biochem. Biophys. Res. Commun. 146: 470-477(1987).

19. Rosenberg, A., et al., Gene 56: 125-135 (1987).

20. Sanger, F., et al., Proc Nat. Acad. Sci. 74: 5463-5467 (1977).

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22. Seno, M., et al., Eur. Pat. Ap. Pub. No. 326,907 (1989).

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24. Thomas Jnr. K. A. and Linemeyer, D. L., Eur. Pat. Ap. Pub. No.319,052 (1989).

25. Walicke, P., et al., Proc. Nat. acad. Sci. USA 83: 3012-3016 (1986).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL:                                                           (iv) ANTI-SENSE:                                                               (v) FRAGMENT TYPE:                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (B) STRAIN:                                                                   (C) INDIVIDUAL ISOLATE:                                                       (D) DEVELOPMENTAL STAGE:                                                      (E) HAPLOTYPE:                                                                (F) TISSUE TYPE:                                                              (G) CELL TYPE:                                                                (H) CELL LINE:                                                                (I) ORGANELLE:                                                                (vii) IMMEDIATE SOURCE:                                                       (A ) LIBRARY:                                                                 (B) CLONE:                                                                    (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT:                                                       (B) MAP POSITION:                                                             (C) UNITS:                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:                                                                   (B) TITLE:                                                                   (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO:                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ATGGCTGAAGGGGAAATC ACCACGCTGCCCGCCCTTCCG39                                    MetAlaGluGlyGluIleThrThrLeuProAlaLeuPro                                       1510                                                                          GAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGGCACTTC78                                     GluAspGly GlySerGlyAlaPheProProGlyHisPhe                                      152025                                                                        AAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTC117                                    LysAspProLysArgLeuTyrCysLysAsnGlyGlyP he                                      3035                                                                          TTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTC156                                    PheLeuArgIleHisProAspGlyArgValAspGlyVal                                       404550                                                                         CGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAA195                                   ArgGluLysSerAspProHisIleLysLeuGlnLeuGln                                       556065                                                                        GCAGAAGAGAGAGGAGTTGTGTCTA TCAAAGGAGTGTGT234                                   AlaGluGluArgGlyValValSerIleLysGlyValCys                                       7075                                                                          GCTAACCGGTACCTGGCTATGAAGGAAGATGGAAGATTA273                                    AlaAsnArgTyrLeu AlaMetLysGluAspGlyArgLeu                                      808590                                                                        CTGGCTTCTAAATGTGTTACGGATGAGTGTTTCTTTTTT312                                    LeuAlaSerLysCysValThrAspGluCysPhePhePhe                                        95100                                                                        GAACGATTGGAATCTAATAACTACAATACTTACCGGTCT351                                    GluArgLeuGluSerAsnAsnTyrAsnThrTyrArgSer                                       105110115                                                                     AGAAAAT ACACCAGTTGGTATGTGGCATTGAAACGAACT390                                   ArgLysTyrThrSerTrpTyrValAlaLeuLysArgThr                                       120125130                                                                     GGGCAGTATAAACTTGGTTCCAAAACAGGACCT GGGCAG429                                   GlyGlnTyrLysLeuGlySerLysThrGlyProGlyGln                                       135140                                                                        AAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGC465                                       LysAlaIleLeuPheLeuProM etSerAlaLysSer                                         145150155                                                                     (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL:                                                           (iv ) ANTI-SENSE:                                                             (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM:                                                                 (B) STRAIN:                                                                   (C) INDIVIDUAL ISOLATE:                                                       (D) DEVELOPMENTAL STAGE:                                                      (E) HAPLOTYPE:                                                                (F) TISSUE TYPE:                                                              (G) CELL TYPE:                                                                (H) CELL LINE:                                                                (I) ORGANELLE:                                                                (vii) IMMEDIATE SOURCE:                                                        (A) LIBRARY:                                                                 (B) CLONE:                                                                    (viii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT:                                                       (B) MAP POSITION:                                                             (C) UNITS:                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY:                                                                 (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD:                                                    (D) OTHER INFORMATION:                                                        (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:                                                                   (B) TITLE:                                                                   (C) JOURNAL:                                                                  (D) VOLUME:                                                                   (E) ISSUE:                                                                    (F) PAGES:                                                                    (G) DATE:                                                                     (H) DOCUMENT NUMBER:                                                          (I) FILING DATE:                                                              (J) PUBLICATION DATE:                                                         (K) RELEVANT RESIDUES IN SEQ ID NO:                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      ATGGCTGAAGGGGAA ATCACCACGCTGCCCGCCCTTCCG39                                    MetAlaGluGlyGluIleThrThrLeuProAlaLeuPro                                       510                                                                           GAGGATGGCGGCAGCGGCGCCTTCCCGCCCGGGCACTTC78                                     GluAsp GlyGlySerGlyAlaPheProProGlyHisPhe                                      152025                                                                        AAGGACCCCAAGCGGCTGTACTGCAAAAACGGGGGCTTC117                                    LysAspProLysArgLeuTyrCysLysAsnGlyG lnPhe                                      3035                                                                          TTCCTGCGCATCCACCCCGACGGCCGAGTTGACGGGGTC156                                    PheLeuArgIleHisProAspGlyArgValAspGlyVal                                       4045 50                                                                       CGGGAGAAGAGCGACCCTCACATCAAGCTACAACTTCAA195                                    ArgGluLysSerAspProHisIleLysLeuGlnLeuGln                                       556065                                                                        GCAGAAGAGAGAGGAGTTGTGT CTATCAAAGGAGTGTCT234                                   AlaGluGluArgGlyValValSerIleLysGlyValSer                                       7075                                                                          GCTAACCGGTACCTGGCTATGAAGGAAGATGGAAGATTA273                                    AlaAsnArgTyr LeuAlaMetLysGluAspGlyArgLeu                                      808590                                                                        CTGGCTTCTAAATCTGTTACGGATGAGTGTTTCTTTTTT312                                    LeuAlaSerLysSerValThrAspGluCysPhePhePhe                                        95100                                                                        GAACGATTGGAATCTAATAACTACAATACTTACCGGTCT351                                    GluArgLeuGluSerAsnAsnTyrAsnThrTyrArgSer                                       105110115                                                                     AGAA AATACACCAGTTGGTATGTGGCATTGAAACGAACT390                                   ArgLysTyrThrSerTrpTyrValAlaLeuLysArgThr                                       120125130                                                                     GGGCAGTATAAACTTGGTTCCAAAACAGGA CCTGGGCAG429                                   GlyGlnTyrLysLeuGlySerLysThrGlyProGlyGlr                                       135140                                                                        AAAGCTATACTTTTTCTTCCAATGTCTGCTAAGAGC465                                       LysAlaIleLeuPheLeuP roMetSerAlaLysSer                                         145150155                                                                 

What is claimed is:
 1. A recombinant chimeric basic fibroblast growthfactor selected from the group consisting of:(a) a chimeric basicfibroblast growth factor having about 155 amino acids wherein thealanine at position 3 and the serine at position 5 are replaced withglutamic acid; (b) a chimeric basic fibroblast growth factor havingabout 155 amino acids wherein the alanine at position 3 and the serineat position 5 are replaced with glutamic acid, and the cysteines atpositions 78 and 96 are replaced with an amino acid selected from thegroup consisting of serine, lysine, aspartic acid, glutamic acid,asparagine, glutamine, histidine, isoleucine, leucine, valine,phenylalanine, tyrosine, methionine, threonine, proline, alanine,glycine, arginine and tryptophan; and (c) the chimeric basic fibroblastgrowth factor of (a) or (b) having no N-terminal methionine;wherein theamino acid at positions 78 and 96 are derivatized with a substituentselected from the group consisting of: CH₂ COOH, CH(CO₂ H) (CH₂)_(x)(CO₂ H), CH₂ CONR₃ R₄, R₅, (CH₂)_(n) SO₃, CHCH₂ CONR₃ CO(CH₂)_(m) NR₃R₄, CH₂ OCOCH₂ R₅ and SR₆ ; wherein R₃ and R₄ are each H, (CH₂)_(x) CO₂H, CHCO₂ H(CH₂)_(x) CO₂ H or C₁ -C₆ alkyl optionally substituted withfrom 0 to 2 hydroxyl groups or polyethylene glycol; R₅ is C₁ -C₆ alkylor C₁ -C₄ alkoxymethyl and R₆ is C₁ -C₆ alkyl, polyethylene glycol orphenyl optionally substituted with one or two carboxylic acid orsulfuric acid groups; n is an integer of from 0 to 4; m is an integer offrom 2 to 4; and x is an integer of from 1 to
 3. 2. The recombinantchimeric basic fibroblast growth factor according to claim 1, whereinsaid chimeric basic fibroblast growth factor is derivatized withcarboxymethyl.
 3. The recombinant chimeric basic fibroblast growthfactor according to claim 2, wherein said fibroblast growth factor isglu³,5 hbFGF.